The present invention relates to novel enzymes that act as enoyl reductases. Two distinct families of enoyl reductases have been identified in bacteria, each of which have a consensus amino acid sequence. The enoyl reductases can be used as targets for designing both new prophylactics and treatments for bacterial infections. Nucleic acid and amino acid sequences of the novel enoyl reductases are also provided.
Essentially all living organisms synthesize saturated fatty acids by the same biochemical mechanism. However, whereas vertebrates and yeast synthesize saturated fatty acids using either one or two multifunctional enzymes (i.e., type I fatty acid synthases, FASs), with the acyl carrier protein (ACP) being an integral part of the complex, most bacteria and plants synthesize saturated fatty acids through the use of a set of distinct enzymes that are each encoded by an individual gene (i.e., type II FASs). In the type II FAS system, ACP is also a distinct protein.
The initial step in the biosynthetic cycle of saturated fatty acids is performed by the enzyme FabH [Tsay et al., J. Biol. Chem. 267:6807-68014 (1992), and U.S. Pat. No: 5,759,832, Issued Jun. 2, 1998, both of which are hereby incorporated by reference in their entireties] which catalyzes the condensation of malonyl-ACP with acetyl-COA. Malonyl-ACP is condensed with the growing-chain acyl-ACP in subsequent rounds by FabB synthase I or by FabF, synthase II. The next step is a ketoester reduction that is catalyzed by an NADPH-dependent xcex2-ketoacyl-ACP reductase (FabG). A xcex2-hydroxyacyl-ACP dehydrase (FabA, dehydrase I or FabZ, dehydrase II) catalyzes the subsequent dehydration forming trans-2-enoyl-ACP. FabI, an NADH-dependent enoyl-ACP reductase, then catalyzes the conversion of trans-2-enoyl-ACP to acyl-ACP to complete the elongation cycle. The addition of two carbon atoms per elongation cycle continues until palmitoyl-ACP is synthesized. Palmitoyl-ACP is one end-product of the pathway and acts as a feedback inhibitor for both FabH and FabI [Heath, et al, J.Biol. Chem. 271:1833-1836 (1996)].
Since an enoyl-ACP reductase catalyzes the final step in the biosynthetic pathway of saturated fatty acids, it is not surprising that it is also a key regulatory target for the pathway [Heath, and Rock, J.Biol.Chem. 271:1833-1836 (1996); Heath and Rock, J.Biol.Chem. 271:10996-11000 [(1996)]. Thus, pharmaceutical companies have placed considerable effort toward developing drugs that inhibit enoyl-ACP reductases and/or the reactions they catalyze. For example, the enoyl-ACP reductase of Mycobacterium tuberculosis (InhA) is a target for the drug isonaizid [Banerjee et al., Science, 263:227 (1994)] whereas, both diazaborines [Baldock et al., Biochem. Phartmacol., 55:1541 (1998)] and triclosan [McMurray et al., Nature (London), 394:531 (1998) and Heath et al., J. Biol. Chem., 273:30316 (1998)] inhibit the Escherichia coli enoyl-ACP reductase, FabI. All three drugs act through the formation of a high-affinity enzyme-NAD+-drug ternary complex [Heath et al., J. Biol. Chem., 274:11110-11114 (1999) and Rozwarski et al., Science, 279:98 (1998); Baldock et al., Science, 274:2107 (1996); Levy et al., Nature (London) 398:383 (1999); Stewart et al, J. Mol. Biol., 290:859 (1999); and Ward et al., Biochemistry, 38:12514 (1999)]. Consistently, missense mutations resulting in single arnino acid substitutions in the active sites of the enoyl-ACP reductases prevent the formation of the ternary complexes and confer a resistant phenotype to bacteria expressing the mutant proteins [Banerjee et al., Science, 263:227 (1994); McMurray et al., Nature (London), 394:531 (1998); Heath et al., J. Biol. Chem., 273:30316 (1998); Heath et al., J. Biol. Chem., 274:11110-11114 (1999); and Bergler et al., J. Gen. Microbiol., 138:2093 (1992) and Rouse et al., Antimicrobiol. Agents. Chem., 39:2472 (1995)].
Unfortunately, the toxicity of boron severely limits the pharmaceutical application of diazaborines [Baldock et al, Biochem. Phannacol., 55:1541(1998)]. Triclosan, on the other hand, is widely employed as an antibacterial in consumer products for external use. Triclosan is a diphenyl ether (bis-phenyl) derivative, known as either 2,4,4xe2x80x2-Trichloro-2xe2x80x2-hydroxydiphenyl ether or 5-Chloro-2-(2,4-dichlorophenoxy) phenol, and is used as an antibacterial in antimicrobial creams, antiperspirants, body washes, cosmetics, deodorants, deodorant soaps, detergents, dish washing liquids, hand soaps, lotions, and toothpaste, as well as in plastics, polymers and textiles [see, Bhargava and Leonard, Am. J. Infect. Control, 24:209 (1996)]. However, the hydrophobic nature and chlorine content of triclosan makes it undesirable for internal use.
Bacterial infections remain among the most common and deadly causes of human disease. For example, Streptococci are known to cause otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis. In addition, virulent strains of E. coli can cause severe diarrhea, a condition which worldwide kills a million more people (3 million) every year than malaria [D. Leff, BIOWORLD TODAY, 9:1,3 (1998)]. Indeed, infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide [Binder et al., Science 284:1311-1313 (1999)].
Although, there was initial optimism in the middle of the 20th century that diseases caused by bacteria would be quickly eradicated, it has become evident that the so-called xe2x80x9cmiracle drugsxe2x80x9d are not sufficient to accomplish this task. Indeed, antibiotic resistant pathogenic strains of bacteria have become common-place, and bacterial resistance to the new variations of these drugs appears to be outpacing the ability of scientists to develop effective chemical analogs of the existing drugs [See, Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998)]. Therefore, new approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
Classical penicillin-type antibiotics effect a single class of proteins known as autolysins. Therefore, the development of new drugs which effect an alternative bacterial target protein would be desirable. Such a target protein ideally would be indispensable for bacterial survival. Thus the identification of a new bacterial enzyme that is required for fatty acid synthesis would be a prime candidate for such drug development.
Therefore, there is a need to identify new proteins that have enzymatic activities that are crucial for bacterial growth. There is also a need to provide immunogenic compositions containing such enzymes or fragments thereof. In addition, there is a need to develop methods for identifying drugs that interfere with such enzymes. Finally, there is a need to employ such procedures to develop new anti-bacterial drugs.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides two families of enzymes that can act as enoyl reductases. One such family shares a common amino acid consensus sequence, SEQ ID NO:45 and binds a flavin cofactor. This family of enoyl reductases is exemplified by the Streptococcus pneumoniae, FabK having an amino acid sequence of SEQ ID NO:2 and is naturally encoded by SEQ ID NO:1, as disclosed herein. The other family of enoyl reductases shares a common amino acid consensus sequence, SEQ ID NO:57 and like the previously disclosed FabI does not contain a flavin cofactor. This second family of enoyl reductases is exemplified by the Campylobacter jejuni FabL having an amino acid sequence of SEQ ID NO:52 and is naturally encoded by SEQ ID NO:51, as disclosed herein.
As disclosed herein and exemplified below, bacteria can express either or both of two unique enoyl reductases, FabK and/or FabL each of which catalyze the identical reaction as the well-characterized Gram-negative bacterial enoyl-ACP reductase, FabI. Since FabI has been a useful target for the design of antibacterials, the identification of FabK and FabL provides another important target. Indeed, the disclosure of FabK and FabL and their related analogs should have a major impact on the development of new prophylactics and treatments for bacterial infections, including those pharmaceuticals that can be used to combat antibiotic resistant Streptococcus and Enterococcus strains. 
Thus the present invention provides an isolated nucleic acid that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:45. The present invention further provides an isolated nucleic acid that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:57. Preferably the polypeptide acts enzymatically as an enoyl reductase. In the case of FabK enoyl reductases, such nucleic acids preferably encode a polypeptide that also binds a flavin prosthetic group. Although the enoyl reductase can be obtained form any source, particularly from fungus, bacteria or plants, in a preferred embodiment the enoyl reductase is not a yeast enzyme. More preferably, the polypeptide is a bacterial enzyme or an active fragment of the bacterial enzyme. The polypeptides encoded by the nucleic acids are also part of the present invention.
In one such embodiment the nucleic acid encodes a bacterial enzyme that comprises an amino acid sequence of SEQ ID NO:2. In another embodiment the nucleic acid encodes a bacterial enzyme that comprises the amino acid sequence of SEQ ID NO:2 comprising a conservative amino acid substitution. In related embodiments, the nucleic acid encodes a bacterial enzyme that comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:6, or the amino acid sequence of SEQ ID NO:6 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:10, or the amino acid sequence of SEQ ID NO:10 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:12, or the amino acid sequence of SEQ ID NO:12 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:14, or the amino acid sequence of SEQ ID NO:14 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:16, or the amino acid sequence of SEQ ID NO:16 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:18, or the amino acid sequence of SEQ ID NO:18 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:20, or the amino acid sequence of SEQ ID NO:20 comprising a conservative amino acid substitution.
In other embodiments the nucleic acid encodes a bacterial enzyme that comprises the amino acid sequence of SEQ ID NO:28 or the amino acid sequence of SEQ ID NO:28 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:30, or the amino acid sequence of SEQ ID NO:30 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:34, or the amino acid sequence of SEQ ID NO:34 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:38, or the amino acid sequence of SEQ ID NO:38 comprising a conservative amino acid substitution. The present invention further provides a nucleic acid encoding a bacterial enzyme that comprises an amino acid sequence of SEQ ID NO:52. In another embodiment the nucleic acid encodes a bacterial enzyme that comprises the amino acid sequence of SEQ ID NO:52 comprising a conservative amino acid substitution. In related embodiments, the nucleic acid encodes a bacterial enzyme that comprises the amino acid sequence of SEQ ID NO:54 or the amino acid sequence of SEQ ID NO:54 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:56, or the amino acid sequence of SEQ ID NO:56 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:50, or the amino acid sequence of SEQ ID NO:50 comprising a conservative amino acid substitution.
In a particular embodiment the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:1. In related embodiments the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:3, or the nucleotide sequence of SEQ ID NO:5, or the nucleotide sequence of SEQ ID NO:9, or the nucleotide sequence of SEQ ID NO:1, or the nucleotide sequence of SEQ ID NO:13, or the nucleotide sequence of SEQ ID NO:15, or the nucleotide sequence of SEQ ID NO:17, or the nucleotide sequence of SEQ ID NO:19.
Still other related embodiments comprise the nucleotide sequence of SEQ ID NO:27, or the nucleotide sequence of SEQ ID NO:29, or the nucleotide sequence of SEQ ID NO:33, or the nucleotide sequence of SEQ ID NO:37.
In a particular embodiment the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:51. In related embodiments the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO:53, or the nucleotide sequence of SEQ ID NO:55, or the nucleotide sequence of SEQ ID NO:49.
The polypeptides encoded by all of the novel nucleic acids disclosed above are also part of the present invention.
The present invention also includes an isolated nucleic acid that hybridizes under standard hybridization conditions to a nucleic acid (e.g., a cDNA) comprising one or more of the nucleotide sequences of the present invention. In a preferred embodiment the isolated nucleic acid hybridizes to the nucleotide sequence of SEQ ID NO:1. In another preferred embodiment the isolated nucleic acid hybridizes to the nucleotide sequence of SEQ ID NO:51. In related embodiments, the isolated nucleic acid hybridizes to the nucleotide sequence of SEQ ID NO:3, and/or the nucleotide sequence of SEQ ID NO:5, and/or the nucleotide sequence of SEQ ID NO:9, and/or the nucleotide sequence of SEQ ID NO:11, and/or the nucleotide sequence of SEQ ID NO:13, and/or the nucleotide sequence of SEQ ID NO:15, and/or the nucleotide sequence of SEQ ID NO:17, and/or the nucleotide sequence of SEQ ID NO:19. In still other related embodiments the isolated nucleic acid hybridizes to the nucleotide sequence of the nucleotide sequence of SEQ ID NO:53, and/or the nucleotide sequence of SEQ ID NO:55, and/or the nucleotide sequence of SEQ ID NO:49.
Such nucleic acids include those that can act as probes or primers for one or more of the nucleotide sequences of the present invention. The polypeptides encoded by the novel nucleic acids that hybridize to the nucleic acids described above are also part of the present invention.
The present invention further provides a recombinant DNA molecule that comprises an isolated nucleic acid of the present invention, as described above with or without a heterologous nucleotide sequence. Such a recombinant DNA molecule can be operatively linked to an expression control sequence and can be part of an expression vector. The present invention further provides a cell that comprises such an expression vector. The cell can be either a eukaryotic or preferably a prokaryotic cell. The present invention further provides a method of expressing a recombinant polypeptide of the present invention or fragment thereof in this cell. One such method comprises culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the polypeptide by the cell. Preferably the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO:45, can bind a flavin prosthetic group, and can act enzymatically as an enoyl reductase. In an alternative embodiment the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO:57, does not contain a flavin prosthetic group, and can act enzymatically as an enoyl reductase. In a preferred embodiment the method comprises the step of purifying the recombinant polypeptide. The recombinant polypeptide purified by the method is also part of the present invention.
The present invention further provides a nucleic acid that encodes a polypeptide that binds a flavin prosthetic group, has enoyl reductase activity and has at least 30%, preferably 60%, more preferably 75%, even more preferably 90% and most preferably 95% amino acid identity with a bacterial enzyme comprising the amino acid sequence of SEQ ID NO:2. In a preferred embodiment the nucleic acid encodes a FabK. In related embodiments, the nucleic acid encodes a polypeptide that binds a flavin prosthetic group, has enoyl reductase activity and has at least 60%, preferably 80%, and more preferably 90% amino acid identity with a bacterial enzyme comprising the amino acid sequence of SEQ ID NO:4, and/or the amino acid sequence of SEQ ID NO:6, and/or the amino acid sequence of SEQ ID NO:10, and/or the amino acid sequence of SEQ ID NO:12, and/or the amino acid sequence of SEQ ID NO:14, and/or the amino acid sequence of SEQ ID NO:16, and/or the amino acid sequence of SEQ ID NO:18, and/or the amino acid sequence of SEQ ID NO:20. Again in preferred embodiments the nucleic acid encodes a FabK. The polypeptides encoded by the nucleic acids described above are also part of the present invention.
The present invention also provides a nucleic acid that encodes a polypeptide that does not contain a flavin prosthetic group, has enoyl reductase activity and has at least 40%, preferably 75%, more preferably 85%, even more preferably 90% and most preferably 95% amino acid identity with a bacterial enzyme comprising the amino acid sequence of SEQ ID NO:52. The polypeptides encoded by the nucleic acids described above are also part of the present invention.
The present invention also provides a nucleic acid that encodes a polypeptide that binds a flavin prosthetic group, has enoyl reductase activity and that comprises at least 8, preferably 12 and more preferably 16 consecutive amino acids of a bacterial enzyme that has an amino acid sequence of SEQ ID NO:2. In a preferred embodiment the nucleic acid encodes a FabK. In related embodiments, the nucleic acid encodes a polypeptide that binds a flavin prosthetic group, has enoyl reductase activity and comprises at least 8, preferably 12 and more preferably 16 consecutive amino acids of a bacterial enzyme that has an amino acid sequence of SEQ ID NO:4, and/or the amino acid sequence of SEQ ID NO:6, and/or the amino acid sequence of SEQ ID NO:10, and/or the amino acid sequence of SEQ ID NO:12, and/or the amino acid sequence of SEQ ID NO:14, and/or the amino acid sequence of SEQ ID NO:16, and/or the amino acid sequence of SEQ ID NO:18, and/or the amino acid sequence of SEQ ID NO:20. In preferred embodiments the nucleic acid encodes a FabK. The polypeptides encoded by the nucleic acids described above are also part of the present invention.
The present invention further provides a nucleic acid that encodes a polypeptide that does not contain a flavin prosthetic group, has enoyl reductase activity and that comprises at least 8, preferably 12 and more preferably 16 consecutive amino acids of a bacterial enzyme that has an amino acid sequence of SEQ ID NO:52. In a preferred embodiment the nucleic acid encodes a FabL. In related embodiments, the nucleic acid encodes a polypeptide that does not contain a flavin prosthetic group, has enoyl reductase activity and comprises at least 8, preferably l2 and more preferably 16 consecutive amino acids of a bacterial enzyme that has an amino acid sequence of SEQ ID NO:54, and/or the amino acid sequence of SEQ ID NO:56, and/or the amino acid sequence of SEQ ID NO:50. The polypeptides encoded by the nucleic acids described above are also part of the present invention.
The present invention further provides fragments of the polypeptides of the present invention and fusion proteins/peptides including chimeric proteins and intein fusion proteins/peptides. The fusion proteins/peptides can comprise any of the polypeptides of the present invention including the fragments of the polypeptides. Such fragments include antigenic fragments, proteolytic fragments, such as peptides prepared by treatment with a protease e.g., trypsin, active fragments that retain enoyl reductase activity, and peptides comprising at least 5, preferably 12 and more preferably 20 consecutive amino acids of a bacterial enzyme that has the amino acid sequence of SEQ ID NO:2 and/or the amino acid sequence of SEQ ID NO:4, and/or the amino acid sequence of SEQ ID NO:6, and/or the amino acid sequence of SEQ ID NO:10, and/or the amino acid sequence of SEQ ID NO:12, and/or the amino acid sequence of SEQ ID NO:14, and/or the amino acid sequence of SEQ ID NO:16, and/or the amino acid sequence of SEQ ID NO:18, and/or the amino acid sequence of SEQ ID NO:20. In a particular embodiment, the antigenic fragment comprises the amino acid sequence of SEQ ID NO:46 or SEQ ID NO:46 comprising a conservative amino acid substitution.
In a related embodiment such fragments comprise at least 5, preferably 12 and more preferably 20 consecutive amino acids of a bacterial enzyme that has the amino acid sequence has the amino acid sequence of SEQ ID NO:52 and/or the amino acid sequence of SEQ ID NO:54, and/or the amino acid sequence of SEQ ID NO:56, and/or the amino acid sequence of SEQ ID NO:50. In a particular embodiment, the antigenic fragment comprises the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:58 comprising a conservative amino acid substitution.
The present invention also provides fragments and fusion proteins/peptides as defined above for the enoyl reductases having the amino acid sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:34, and SEQ ID NO:38.
In addition, the present invention provides proteins and fragments and fusion proteins/peptides as defined above having the amino acid sequences of SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:36 and SEQ ID NO:48.
The present invention also provides immunogenic compositions and vaccines. In a particular embodiment the vaccine comprises an antigenic fragment of the present invention. Antibodies to the enoyl reductases and antigenic fragments of the present invention are also included. Such antibodies can be monoclonal antibodies, and/or chimeric antibodies or polyclonal antibodies. The present invention further provides an immortal cell line that produces a monoclonal antibody of the present invention. In a particular embodiment, the monoclonal antibody is raised against a polypeptide or fragment thereof comprising SEQ ID NO:46. In another embodiment, the monoclonal antibody is raised against a polypeptide or fragment thereof comprising SEQ ID NO:58.
The present invention further provides methods for identifying agents that can modulate the enzymatic activity of an enoyl reductase of the present invention. One such embodiment comprises measuring the enzymatic activity of an enoyl reductase or active fragment thereof in the presence and absence of a compound. The compound is identified as an agent that modulates the enzymatic activity of an enoyl reductase when the enzymatic activity measured is different in the presence of the compound relative to in the absence of the compound. In a preferred embodiment of this type, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:45 and contains a flavin prosthetic group. In another preferred embodiment of this type, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:57 and does not contain a flavin prosthetic group. In a particular embodiment, the enzymatic activity is lower in the presence of the compound relative to in the absence of the compound. In this case the compound is identified as an inhibitor of the enoyl reductase. In another embodiment of this type, the enzymatic activity is higher in the presence of the compound relative to in the absence of the compound. In this case the compound is identified as an agonist of the enoyl reductase. In one particular embodiment, the enoyl reductase is a FabK. In another particular embodiment, the enoyl reductase is a FabL.
The present invention further provides methods for identifying an agent that can bind to an enoyl reductase. One such embodiment comprises contacting an enoyl reductase or active fragment thereof with a compound and determining whether the compound binds to the enoyl reductase. A compound is identified as an agent that can bind the enoyl reductase if the compound binds to the enoyl reductase. In a preferred embodiment of this type, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:45 and contains a flavin prosthetic group. In another preferred embodiment of this type, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:57 and does not contain a flavin prosthetic group.
In the in vitro studies involving the enoyl reductases of the present invention, the enoyl reductase preferably has the amino acid sequence of SEQ ID NO:2. However, in other embodiments of the present invention, the enoyl reductase has the amino acid sequence of SEQ ID NO:2 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:4, or the amino acid sequence of SEQ ID NO:4 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:6, or the amino acid sequence of SEQ ID NO:6 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO10, or the amino acid sequence of SEQ ID NO:10 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:12, or the amino acid sequence of SEQ ID NO:12 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:14, or the amino acid sequence of SEQ ID NO:14 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:16, or the amino acid sequence of SEQ ID NO:16 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:18, or the amino acid sequence of SEQ ID NO:18 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:20, or the amino acid sequence of SEQ ID NO:20 comprising a conservative amino acid substitution SEQ ID NO:22.
Similarly, the enoyl reductase can comprise the amino acid sequence of SEQ ID NO:52. In another embodiment the enoyl reductase comprises the amino acid sequence of SEQ ID NO:52 comprising a conservative amino acid substitution. In related embodiments, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:54 or the amino acid sequence of SEQ ID NO:54 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:56, or the amino acid sequence of SEQ ID NO:56 comprising a conservative amino acid substitution, or the amino acid sequence of SEQ ID NO:50, or the amino acid sequence of SEQ ID NO:50 comprising a conservative amino acid substitution.
As mentioned above, fusion proteins/peptides and/or fragments, and preferably active fragments of the enoyl reductases can also be used.
The present invention further provides methods for identifying a drug that inhibits bacterial growth. One such embodiment comprises administering an agent that is suspected of inhibiting an enoyl reductase of the present invention to a bacterial cell and then determining the growth of the cell. An agent that inhibits the growth of the cell relative to the growth in the absence of the agent is identified as a drug that inhibits bacterial growth. In a preferred embodiment of this type, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:45 and contains a flavin prosthetic group. In a more preferred embodiment, the enoyl reductase is a FabK. Alternatively, the enoyl reductase comprises the amino acid sequence of SEQ ID NO:57 and does not contain a flavin prosthetic group. In a more preferred embodiment of this type, the enoyl reductase is a FabL.
As should be readily understood, any of the methods described above can be performed either alone, or in tandem including the combination of two or more of the above-described methods. For example, an agent could be first tested for binding, then tested for inhibiting the enoyl reductase. An agent that both binds the enoyl reductase and inhibits the enoyl reductase activity could then be tested to determine if it also inhibited bacterial cell growth. Further studies could be performed in an animal model to determine if the agent was effective in either preventing or treating a bacterial infection. An agent found to be effective in an animal model could then be used in a clinical study. Thus the present invention further provides the agents and drugs identified by the methods of the present invention and the corresponding pharmaceutical compositions, which can further comprise a pharmaceutically acceptable carrier.
Accordingly, it is a principal object of the present invention to provide novel enoyl reductases. Such enzymes can used as targets in drug discovery including for high throughput screening and/or rational drug design.
It is a further object of the present invention to provide methods of using these enoyl reductases to identify agents that will act against bacterial infections.
It is a further object of the present invention to provide antibacterial agents obtained by the methods of the present invention.
It is a further object of the present invention to provide structural and enzymatic characteristics and properties of the enoyl reductases, including their nucleic acid and amino acid sequences.
It is a further object of the present invention to provide an antibody specific for FabK.
It is a further object of the present invention to provide an antibody specific for FabL.
It is a further object of the present invention to provide an immunogenic composition comprising a FabK, or an antigenic fragment of FabK.
It is a further object of the present invention to provide a vaccine comprising a nucleic acid encoding a FabK or an antigenic fragment of a FabK.
It is a further object of the present invention to provide an immunogenic composition comprising a FabK, or an antigenic fragment of FabL
It is a further object of the present invention to provide a vaccine comprising a nucleic acid encoding a FabK or an antigenic fragment of a FabL.
It is a further object of the present invention to provide a method of producing an enoyl reductase of the present invention, including through modification or fragmentation of an enoyl reductase through recombinant technology.
It is a further object of the present invention to provide a method of performing rational drug design with the use of an enoyl reductase of the present invention.